Predicting the 3D structure of protein interactions remains a challenge in the field of computational structural biology. This is in part due to difficulties in sampling the complex energy landscape of multiple interacting flexible polypeptide chains. Coarse-graining approaches, which reduce the number of degrees of freedom of the system, help address this limitation by smoothing the energy landscape, allowing an easier identification of the global energy minimum. They also accelerate the calculations, allowing to model larger assemblies. Here, we present the implementation of the MARTINI coarse-grained force field for proteins into HADDOCK, our integrative modelling platform. Docking and refinement are performed at the coarse-grained level and the resulting models are then converted back to atomistic resolution through a distance restraints-guided morphing procedure. Our protocol, tested on the largest complexes of the protein docking benchmark 5, shows an overall ~7-fold speed increase compared to standard all-atom calculations, while maintaining a similar accuracy and yielding substantially more near-native solutions. To showcase the potential of our method, we performed simultaneous 7 body docking to model the 1:6 KaiC-KaiB complex, integrating mutagenesis and hydrogen/deuterium exchange data from mass spectrometry with symmetry restraints, and validated the resulting models against a recently published cryo-EM structure.

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein–protein docking to investigate the conformational changes necessary for the formation of the CPR–CYP complex. While some structural features of the CPR and the CPR–CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR–CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR–CYP interactions and underlying conformational changes, improving our understanding of this complex machinery relevant for drug metabolism.

Characterization of cytochrome P450 2D6 (CYP2D6) and the impact of the major identified allelic variants on the activity of one of the most dominating drug-metabolising enzymes is essential to increase drug safety and avoid adverse reactions. Microsecond molecular dynamics simulations have been performed to capture the dynamic signatures of this complex enzyme and five allelic variants with diverse enzymatic activity. In addition to the apo simulations, three substrates (bufuralol, veliparib and tamoxifen) and two inhibitors (prinomastat and quinidine) were included to explore their influence on the structure and dynamical features of the enzyme. Our results indicate that the altered enzyme activity can be attributed to changes in the hydrogen bonding network within the active site, and local structural differences in flexibility, position and shape of the binding pocket. In particular, the increased (CYP2D6*53) or the decreased (CYP2D6*17) activity seems to be related to a change in dynamics of mainly the BC loop due to a modified hydrogen bonding network around this region. In addition, the smallest active site volume was found for CYP2D6*4 (no activity). CYP2D6*2 (normal activity) showed no major differences in dynamic behaviour compared to the wild-type.